In U.S. Pat. No. 5,047,321, Loken and Terstappen described the multi-parameter analysis of cellular components in a body fluid. The body fluids described included blood and bone marrow. Using a combination of two nucleic acid dyes, a fluorescently labelled monoclonal antibody and two light scatter parameters, Loken and Terstappen were able to discriminate between and among various components of blood and bone marrow, count the number of cells within each component and provide a differential analysis of each.
Loken and Terstappen used LDS-751 (Exciton) as a DNA dye, Thiazole Orange ("TO", Molecular Probes, Inc.) as an RNA dye, a fluorescently labelled anti-CD45 monoclonal antibody and forward and orthogonal light scatter on whole blood or bone marrow aspirates. Using these five parameters, they were able to detect and differentiate between erythrocytes, reticulocytes, nucleated erythrocytes, platelets, lymphocytes, monocytes, neutrophilic granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells.
Specifically, erythrocytes were characterized by light scatter and lack of fluorescence. Reticulocytes were characterized similar to erythrocytes by light scatter but could be discriminated from erythrocytes based upon their staining with Thiazole Orange. Platelets were characterized by their low light scatter and staining with LDS-751. Leukocytes were characterized by their large light scatter, LDS-751 and Thiazole Orange fluorescence and anti-CD45 fluorescence. Among the leukocytes, lymphocytes were characterized by high fluorescence intensity of anti-CD45 staining; monocytes had similar antibody fluorescence intensity with larger light scatter; neutrophilic granulocytes had dimmer antibody fluorescence intensity with large light scatter; and eosinophilic granulocytes had an antibody fluorescence intensity similar to monocytes but had a larger orthogonal light scatter and lower forward light scatter than monocytes.
While the method has utility for most analyses, a limitation in this method exists. The combination of LDS-751 with Thiazole Orange and anti-CD45 fluorescence does not permit full discrimination among the erythroid lineage (i.e., it does not permit identification of orthochromatic normoblasts, normoblasts and erythroblasts and does not permit differentiation between mature and immature reticulocytes) and does not permit the identification of proliferating myeloid cells and non-hematopoietic cells (i.e., stromal and epithelial cells).
Taking these limitations into account, there is a need for an improved method for the identification of and discrimination among the cellular components of blood and bone marrow.